HAL Id: hal-02339785
https://hal.sorbonne-universite.fr/hal-02339785
Submitted on 30 Oct 2019
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral
and neuroendocrine responses
Daphné Capela, Kévin Poissenot, Carlos Dombret, Matthieu Keller, Isabelle Franceschini, Sakina Mhaouty-Kodja
To cite this version:
Daphné Capela, Kévin Poissenot, Carlos Dombret, Matthieu Keller, Isabelle Franceschini, et al.. Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphe- nol on behavioral and neuroendocrine responses. Chemosphere, Elsevier, 2019, 221, pp.573-582.
�10.1016/j.chemosphere.2019.01.071�. �hal-02339785�
Title 1
Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on 2
behavioral and neuroendocrine responses 3
Running title 4
Effects of co-exposure to DEHP/NP in male mice 5
Authors 6
Daphné Capela
1*, Kevin Poissenot
2*, Carlos Dombret
1, Matthieu Keller
2, Isabelle Franceschini
2, 7
Sakina Mhaouty-Kodja
18
*Equal contribution 9
Affiliations 10
1
Sorbonne Université, CNRS, INSERM, Neuroscience Paris Seine – Institut de Biologie Paris-Seine, 11
75005 Paris, France.
12
2
UMR Physiologie de la Reproduction & des Comportements, Institut National de la Recherche 13
Agronomique, Centre National de la Recherche Scientifique, Université de Tours, Institut Français du 14
Cheval et de l’Equitation, Nouzilly 37380, France.
15 16
Corresponding authors 17
Sakina Mhaouty-Kodja 18
Sorbonne Université, CNRS UMR 8246, INSERM U1130 19
7 quai St Bernard, Bât A 3ème étage 75005, Paris, France.
20
Tel: +331 44 27 91 38 21
sakina.mhaouty-kodja@sorbonne-universite.fr 22
23
Abstract 24
The present study evaluates the effects of adult exposure to low doses of a mixture of di-(2- 25
ethylexyl)phthalate (DEHP) and nonylphenol (NP) on reproductive neuroendocrine function and 26
behavior. The neural circuitry that processes male sexual behavior is tightly regulated by testosterone 27
and its neural metabolite estradiol. In previous studies, we showed that adult exposure of mice to low 28
doses of each of these widespread environmental contaminants resulted in altered sexual behavior, 29
without any effect on the regulation of the gonadotropic axis. Here, adult C57BL/6J male mice were 30
exposed to DEHP/NP (0.5 or 5 μg/kg body weight/day) for 4 weeks before starting the analyses. Mice 31
treated with DEHP/NP at 0.5 μg/kg/day show altered olfactory preference, and fewer of them emit 32
ultrasonic vocalization compared to the other treatment groups. These mice also exhibit a lower 33
number of mounts and thrusts, increased locomotor activity and unaffected anxiety-state level, along 34
with unaltered testosterone levels and kisspeptin system, a key regulator of the gonadotropic axis.
35
Analysis of the neural circuitry that underlies sexual behavior showed that the number of cells 36
expressing androgen and estrogen receptors is comparable between control and DEHP/NP-exposed 37
males. The comparison of these data with those obtained in males exposed to each molecule separately 38
highlights synergistic effects at the lower dose of contaminants of 0.5 μg/kg/day. In contrast, the 39
effects previously observed for each molecule at 5 μg/kg/day were not detected. A detailed 40
comparison of the effects triggered by separate or combined exposure to DEHP and NP is discussed.
41
42
Highlights 43
Adult co-exposure to DEHP/NP alters male sexual behavior.
44
DEHP/NP mixture increases locomotor activity and reduces anxiety-state level.
45
The lower dose of DEHP/NP mixture triggers the majority of behavioral effects.
46
No effects of DEHP/NP mixture on the gonadotropic axis.
47
Unaltered number of AR- and ERα-immunoreactive cells in exposed males.
48
49
Keywords 50
Nervous system 51
Endocrine disruptor 52
Phthalates 53
Nonylphenol 54
Behavior 55
Reproduction 56
57
1. Introduction 58
Di-(2-ethylexyl)phthalate (DEHP) and nonylphenol (NP) are abundant organic pollutants found in 59
the environment. While DEHP is used in the manufacture and processing of plastic products, NP 60
derives from the degradation of alkylphenol that is used in a wide variety of industrial, agricultural and 61
domestic applications such as soap, cosmetics, paint, herbicides and pesticides, or plastic fabrication.
62
Both DEHP and NP were classified by the EU in 2000 as priority substances “presenting a significant 63
risk to or via the aquatic environment” in the Water Framework Directive 2000/60/EC, which was 64
updated in 2008 and 2013 (Directive 2013/39/EU of the European Parliament and of the Council of 12 65
August 2013). These molecules were described as exhibiting, respectively, estrogenic activity for NP 66
(Laws et al., 2000), and anti-androgenic effects like the reduction of testosterone production for DEHP 67
(Barakat et al., 2017; Pocar et al., 2012).
68
Recently, we showed that chronic treatment of adult male mice with DEHP at 5 or 50 µg/kg/day 69
induced a sexual alteration. This was evidenced by the reduced emission of ultrasonic vocalizations 70
and attractiveness, and a delayed initiation of mating and ejaculation (Dombret et al., 2017). These 71
behavioral modifications were associated with down-regulation of the androgen receptor (AR) in the 72
neural circuitry that underlies sexual behavior, without any changes in the circulating level of 73
testosterone or the integrity of the gonadotropic axis. In contrast, chronic exposure of adult male mice 74
to NP at 5 µg/kg/day increased the emission of ultrasonic vocalizations, the number of mounts, 75
intromissions, and thrusts. This also resulted in delayed ejaculation (Capela et al., 2018). Ten-fold 76
lower and higher doses of NP (0.5 and 50 µg/kg/day, respectively) did not induce any behavioral 77
changes. Once again, exposure to NP altered neither the gonadotropic axis nor testosterone levels.
78
However, it modified the levels of AR and the estrogen receptor (ER) in the neural circuitry that 79
underlies sexual behavior. In this context, whether and how combined exposure to DEHP and NP 80
affects these neural processes, in particular at doses close to that of environmental exposure, remains 81
to be studied.
82
83
In the literature, the few reported studies that address the effects of combined exposure to 84
phthalates and NP focus on peripheral functions. Studies in rats in vitro show a negative additive 85
effect of a mixture containing di-n-butyl phthalate (DBP) or its metabolite mono- butyl phthalate 86
(MBP) and NP. This causes decreased viability of Sertoli cells, and changes in morphological 87
parameters and membrane permeability, as well as increased apoptosis (Hu et al., 2012; Li et al., 88
2010). In another study, the same group observed an alteration in the morphology of tight junctions 89
located between rat Sertoli cells, both in vitro and in vivo, with higher sensitivity to NP than to MBP 90
(Hu et al., 2014a). A similar mixture also resulted in antagonistic effects on levels of testosterone, LH 91
(luteinizing hormone), and FSH (follicle stimulating hormone), at doses ranging from 50 to 450 92
mg/kg/day, indicating competitive interactions between the two chemicals (Hu et al., 2014b). A recent 93
analysis pertaining to combined treatment of mice with phthalates (DEHP, DBP and benzyl butyl 94
phthalate (BBP) at 300 µg/kg/day) and alkylphenols (NP and octylphenols (OP) at 50 µg/kg/day) 95
showed changes in the testicular miRNome, together with decreased testicular estradiol, altered 96
spermatogenesis, and germ cell apoptosis following long-term exposure including gestational, 97
lactational, prepubertal, and pubertal periods (Buñay et al., 2017).
98 99
The present study was undertaken to characterize the effects of chronic exposure of male mice to 100
low doses of the two molecules together. The same behavioral and neuroendocrine endpoints 101
previously tested for each compound alone were analyzed. To this end, male mice were treated orally 102
with the vehicle alone or with DEHP and NP (5 and 0.5 µg/kg/day) for four weeks. Courtship behavior 103
of male mice was analyzed by testing their olfactory preference and emission of courtship vocalization 104
in the presence of receptive females. Latency and frequency of copulatory behavioral events were also 105
quantified. Furthermore, we measured locomotor activity and the anxiety-state level, which, if altered, 106
could interfere with the expression of sexual behavior. Testosterone levels and weight of androgen- 107
sensitive tissues of the genital tract were compared between the three groups. Kisspeptin- 108
immunoreactive neurons were quantified in the rostral preoptic periventricular nucleus (RP3V) and in 109
the arcuate nucleus. Kisspeptin acts as a central regulator of the gonadotropic axis and is currently
110
considered to be a key central target of testosterone feedback regulation for GnRH (gonadotropin- 111
releasing hormone)/LH release (Navarro et al., 2011; Raskin et al., 2009; Ruka et al., 2016; Smith et 112
al., 2005). Finally, the number of AR- and ER-immunoreactive neurons in the neural circuitry that 113
underlies sexual behavior was determined.
114
115
2. Materials and Methods 116
2.1. Animals and treatment 117
Analyses were performed according to European legal requirements (Decree 2010/63/UE) and were 118
approved by the “Charles Darwin” Ethical committee (project number 01490-01). Mice of the 119
C57BL/6J strain (Janvier) were housed in nest-enriched polysulfone cages maintained at 22°C, with a 120
12:12 h light-dark cycle, and were fed a standard diet with free access to food and water. The numbers 121
of experimental and control groups are given in the figure legends.
122
To mimic the major route of exposure to NP and DEHP, eight-week-old mice were fed ad libitum a 123
standard diet containing the vehicle (control group) or DEHP and NP (Sigma-Aldrich). These 124
compounds were dissolved in an ethanol-water mix and were incorporated into the food, so that 125
exposure was equivalent to 0.5 µg/kg body weight/day (DEHP/NP-0.5 group) or 5 µg/kg body 126
weight/day (DEHP/NP-5 group). DEHP and NP doses were calculated for a daily food intake of 5 g 127
per animal. Mice were weighed weekly and DEHP and NP doses adjusted according to changes in 128
their body weight. This latter parameter was followed throughout the whole experimental period, and 129
was similar in the four treatment groups (27 ± 0.3 g, 27.8 ± 0.6 g, 25 ± 0.6 g for the vehicle, 130
DEHP/NP-0.5 and DEHP/NP-5, respectively, on the first day of treatment; and 31 ± 0.6 g, 33.1 ± 0.8 131
g, 32 ± 0.8 g on the last day). Analyses were started after 4 weeks of treatment with DEHP and NP, 132
conditions that were maintained during the whole experimental period.
133 134
2.2. Behavioral tests 135
Tests were conducted under red-light illumination 2 h after lights were turned off, and were 136
videotaped for later analysis as previously described (Capela et al., 2018; Dombret et al., 2017). Four 137
weeks after exposure to DEHP and NP, naïve male mice were housed individually for 3 days and then 138
paired with a sexually receptive female for their first sexual experience as described below (Capela et 139
al., 2018; Dombret et al., 2017). Analyses of recordings were performed by blind observation, since 140
males were identified by numbers attributed at weaning without any information concerning their 141
treatment details.
142
143
2.2.1. Preparation of sexually receptive females 144
C57BL/6J female mice used as stimuli were ovariectomized under general anesthetic 145
(xylazine/ketamine) and implanted with SILASTIC implants (Dow Corning, Midland, MI) filled with 146
50 µg of estradiol-benzoate (Sigma-Aldrich) in 30 µl of sesame oil. Four to 5 h before the tests, 147
females were given a sub-cutaneous injection of 1 mg of progesterone (Sigma-Aldrich) in 100 µl of 148
sesame oil. Female receptivity was verified before the beginning of experiments, using sexually 149
experienced males.
150 151
2.2.2. Olfactory preference test 152
Olfactory preference was assessed in an enclosed plexiglass Y-maze. On the day of the test, male mice 153
were offered the choice between an anesthetized sexually receptive female and an anesthetized 154
gonadally intact male. The time spent in chemo-investigation of each stimulus and the number of 155
entries into each arm of the Y-maze were scored over 10 min. The discrimination index was calculated 156
as the time spent in female investigation (F) minus the time spent in male investigation (M) divided by 157
the total time of investigation (F-M/F+M).
158 159
2.2.3. Ultrasonic vocalization recording 160
Each male mouse was tested in its home cage, in the presence of a sexually receptive female. After 161
introduction of the female into the cage, vocalizations were recorded for 4 min with a microphone 162
(UltraSoundGate) connected to an ultrasound recording interface plugged into a computer equipped 163
with Avisoft-SASLab Pro 5.2.09 recording software, then analyzed using SASLab Pro (Avisoft 164
Bioacoustic). Spectrograms were generated for each call detected (frequency resolution: FFT-length:
165
512; frame size: 100%; overlap: 50%). The parameters used for the automatic quantification of the 166
ultrasonic vocalizations were: cut-off frequency of 30 kHz, element separation based on an automatic 167
single threshold with a hold time of 15 ms. The percentage of animals emitting vocalizations was 168
determined.
169
170
2.2.4. Mating 171
Each male mouse was tested in its home cage for 10 h after introduction of the receptive female. The 172
latency to the first intromission or ejaculation (time from the female introduction into the cage until 173
the behavioral event), the mating duration (time from the first mount until ejaculation), the frequencies 174
of mounts, intromissions and thrusts were scored.
175 176
2.2.5. Locomotor activity 177
Activity was analyzed in a computed circular corridor. Briefly, the male subject was introduced into a 178
circular corridor made up of two concentric cylinders crossed by four diametrically opposite infrared 179
beams (Imetronic). Locomotor activity was counted when animals interrupted two successive beams 180
and had thus traveled a quarter of the circular corridor. Spontaneous activity was recorded for 140 min 181
and was expressed as cumulative activity every 20 min or cumulative activity over the whole 140 min 182
test.
183 184
2.2.6. Anxiety-related behavior 185
Males were placed in the closed arms of the O-maze and were allowed to explore the maze freely for 9 186
min. The latency time before entry into the open arms, the number of entries and the time spent in the 187
open arms were analyzed. A mouse was considered to be in an open arm once all its 4 paws had 188
entered the arm. The light intensity was 60 lux.
189 190
2.3. Immunohistochemistry 191
Animals were euthanized and transcardially perfused with a solution of 4% paraformaldhehyde (PFA) 192
in phosphate buffer. Brains were post-fixed overnight in 4% PFA, cryoprotected in sucrose and stored 193
until analyses. Brains were sliced into coronal sections of 30 µm and immunolabeling processed as 194
performed previously (Capela et al., 2018; Dombret et al., 2017).
195
Kisspeptin immunolabeling was processed with anti-kisspeptin AC053, and sections were 196
counterstained with Hoechst. The number of labeled cell soma and fiber density in the anteroventral 197
periventricular (AVPV) and periventricular nuclei (PeN) of RP3V, and the arcuate nucleus were
198
quantified. Images for fiber were acquired with an LSM 700 confocal microscope (Zeiss) under x40 199
magnification. Quantifications were performed on three sections sampled at one anteroposterior level 200
of RP3V corresponding to the AVPV (plate 29 of Paxinos and Franklin atlas) and at two levels 201
corresponding to the PeN (plates 30 and 31-32), and on three sections of the arcuate nucleus sampled 202
at the levels of the anterior, median and caudal arcuate nuclei (plates 43, 47 and 50).
203
AR- and ERα-immunolabeling were processed as previously described (Capela et al., 2018; Dombret 204
et al., 2017). Images were acquired with a Nikon Eclipse 80i light microscope under x10 205
magnification. The number of labeled cells per section were counted in anatomically matched 206
sections. Counting surfaces for medial amygdala (plate 45 of Paxinos and Franklin atlas), bed nucleus 207
of stria terminalis (plate 33), and medial preoptic nucleus (plate 30) were respectively 0.07 mm
2, 0.055 208
mm
2and 0.31mm
2. 209
210
2.4. Urogenital tract weight and hormone measurements 211
Animals were euthanized to collect their blood and to weigh testes and seminal vesicles. Serum was 212
prepared as previously described (Raskin et al., 2009), and circulating levels of testosterone were 213
measured by RIA using
3H-testosterone at the hormonal assay platform of the laboratory of behavioral 214
and reproductive physiology (UMR INRA/ CNRS / Université de Tours). The mean intra-assay 215
variation coefficient was 7%, and the assay sensitivity was 125 pg/ml.
216 217
2.5. Statistical analyses 218
The percentage of animals emitting ultrasonic vocalizations were compared by chi square Test. The 219
other data were expressed as the mean ± S.E.M. Two-way ANOVA was used to analyze the main 220
effects of treatment and stimulus on olfactory preference (number of entries into the arms), and of 221
treatment and time on locomotor activity. One-way ANOVA was used to analyze the effects of 222
exposure on the remaining data. Tukey post-hoc tests were used to determine group differences. P 223
values of less than 0.05 were considered to be significant.
224
225
3. Results 226
3.1. Effects of adult co-exposure to DEHP and NP on sexual behavior 227
In the presence of a sexually receptive female, the male mouse exhibits olfactory investigation of the 228
female genitals. The pheromonal cues emitted by the female are detected by the olfactory bulb, which 229
transmits signals to the chemosensory areas involved in the activation of male sexual behavior. Male 230
mice exposed to the vehicle or DEHP/NP mixture at 0.5 and 5 µg/kg/day were therefore analyzed for 231
their olfactory preference in a behavioral test using anesthetized gonadally intact males versus 232
sexually receptive females. Figure 1A shows that there was no effect of DEHP/NP exposure on the 233
time spent in total chemo-investigation (p = 0.36). Two-way ANOVA showed no effect of exposure to 234
DEHP/NP (F
(2, 21)= 0.50, p = 0.61) or stimulus (F
(1, 21)= 2.39, p = 0.14) on the number of entries into 235
the Y maze arms (Fig. 1B). In contrast, an effect of DEHP/NP exposure on the ability of mice to 236
discriminate between male and female stimuli was observed (p = 0.0001; Fig. 1C). Indeed, male 237
groups exposed to DEHP/NP at 0.5 µg/kg/day showed no preference for females over males, while 238
such a preference was observed for the vehicle and DEHP/NP-5 groups.
239
Olfactory stimulation activates the emission of ultrasonic vocalization by males (Bean, 1982; Dizinno 240
and Whitney, 1977; Nyby et al., 1977), which seem to play a key role in female attraction and may 241
facilitate copulation (Pomerantz et al., 1983). Male mice mainly vocalized at a frequency of 40-110 242
kHz. The percentage of vocalizing animals differed between the three treatment groups, since only 243
25% of the male group exposed to a DEHP/NP mixture at 0.5 µg/kg/day emitted ultrasonic 244
vocalizations in the presence of sexually receptive females (p = 0.049). In contrast more than 60% of 245
males did so in the two other groups (Fig. 1D).
246
In mating tests, one-way ANOVA showed no significant effect of treatment on the latency to initiate 247
the first mount and first intromission, or time to reaching ejaculation, despite a tendency for these 248
events to be increased in the DEHP/NP-0.5 group (Fig. 1E). Mating duration was also unaffected by 249
exposure to DEHP/NP (Figure 1F). Quantification of the frequency of behavioral events showed an 250
effect of exposure on the number of mounts (p = 0.02) and thrusts (p = 0.03), with males of the
251
DEHP/NP-0.5 group exhibiting less mounts and thrusts than vehicle-exposed mice (Figure 1G). No 252
significant effects on the number of intromissions were observed.
253 254
3.2. General behavioral analysis of males exposed or not to the DEHP/NP mixture 255
General behavior such as locomotor activity or anxiety-state levels were assessed. Two-way ANOVA 256
showed an effect of time (F
(6, 114)= 166.4, p < 0.0001) and exposure (F
(2, 19)= 3.661, p = 0.04) on 257
locomotor activity assessed over 140 min (Fig. 2A). Post hoc analyses showed a higher activity for the 258
DEHP/NP-0.5 group by comparison to males exposed to DEHP/NP-5 at 80 and 100 min of the test. In 259
agreement with this, total activity showed an effect of exposure (p = 0.04), with an increased activity 260
of males treated with 0.5 µg/kg/day of the DEHP/NP mixture (Fig. 2A). In the O-maze test, no effect 261
of exposure on the latency to enter in the open arms was observed (p = 0.33), but an effect of exposure 262
was found on the number of entries (p = 0.02) and on the time spent in the open arms (p = 0.03), as 263
shown in Fig. 2B. Post hoc analyses showed that the DEHP/NP mixture at 5 µg/kg/day induced an 264
anxiolytic behavior as evidenced by the higher number of entries (p < 0.05 versus the vehicle group) 265
and increased time spent in the open arms (p < 0.05 versus the vehicle group), while no effect was 266
seen at the lower dose of 0.5 µg/kg/day.
267
Overall, behavioral data show an effect of exposure to the DEHP/NP mixture on male sexual behavior, 268
including olfactory preference, production of ultrasonic vocalizations, and mating. Effects are seen in 269
particular at the lower dose of pollutants of 0.5 µg/kg/day. The sexual alterations cannot be attributed 270
to changes in general behavior patterns, since males of this group exhibited a higher activity and 271
showed no anxiety-like behavior.
272 273
3.3. Adult co-exposure to a DEHP and NP mixture did not affect the gonadotropic axis 274
Male behaviors are tightly controlled by testosterone. Therefore, DEHP/NP treatment could indirectly 275
induce behavioral modifications through changes in the gonadotropic axis. At the hypothalamic level, 276
we analyzed kisspeptin immunoreactivity in two subdivisions of RP3V (AVPV and PeN subregions) 277
and in the arcuate nucleus. Data illustrated in Figure 3A show the presence of few neurons in the
278
AVPV and PeN subidivisions of RP3V, as is widely known for male mice (Kauffman et al., 2007).
279
One-way ANOVA showed no effect of exposure on kisspeptin immunoreactivity in these 280
hypothalamic areas (Fig. 3A). No differences in fiber density were seen in the arcuate nucleus (Fig.
281
3B). In terms of hormones, we measured circulating levels of testosterone and weights of androgeno- 282
dependent tissues. No effects on circulating levels of testosterone were observed (Fig. 3C). In 283
agreement with these data, the weight of seminal vesicles and testis were comparable between the 284
three treatment groups (Fig. 3D-E). These data show that the DEHP/NP mixtures did not affect the 285
gonadotropic axis at the doses studied.
286 287
3.4. Quantification of AR and ERα immunoreactivity in the neural circuitry underlying sexual 288
behavior 289
Our previous studies showed that chronic exposure to DEHP alone or NP alone altered the number of 290
neurons expressing AR or ERα in the neural circuitry that underlies male sexual behavior (Capela et 291
al., 2018; Dombret et al., 2017). These two receptors play complementary roles in the expression of 292
male behavior (Naulé et al., 2016; Ogawa et al., 1997; Ogawa et al., 1998; Raskin et al., 2009;
293
Wersinger et al., 1997). Immmunohistochemical studies show AR and ERα nuclear staining in the 294
medial amygdala, bed nucleus of stria terminalis, and medial preoptic nuclei (Fig. 4A; 5A). The 295
quantification of AR and ERα-expressing neurons did not show any significant difference between the 296
three treatment groups (Fig. 4B; 5B).
297
Taken together, our data show that exposure to the DEHP/NP mixture at the doses tested did not affect 298
the expression of neural AR or ERα in the neural circuitry that underlies male sexual behavior.
299 300
3.5. Effects of adult exposure to DEHP/NP mixture compared to DEHP and NP alone 301
Table 1 summarizes the effects of the DEHP/NP mixture compared to each compound alone. Several 302
observations can be drawn from such a comparison. Firstly, when males were exposed to each 303
molecule alone, sexual behavior (emission of ultrasonic vocalizations, latency to intromission and 304
ejaculation) or anxiety-state levels were affected at the dose of 5 µg/kg/day, while no effects were
305
observed at a 10-fold lower dose (0.5 µg/kg/day). In the present study, combined exposure induced 306
sexual alterations at the dose of 0.5 µg/kg/day, whereas no behavioral effects were observed at 5 307
µg/kg/day. Secondly, olfactory preferences and locomotor activity were not affected by treatment with 308
each compound alone, whatever the dose used, while the mixture induced effects at the lower dose of 309
0.5 µg/kg/day. Thirdly, while the behavioral effects induced by each compound alone at 5 µg/kg/day 310
were associated with changes in the expression level of AR and ERα in the neural circuitry that 311
underlies sexual behavior, the mixture did not induce any significant modification. Finally, for both 312
types of exposure (to a single molecule or two molecules), kisspeptin-immunoreactivity was unaltered 313
in the hypothalamic nuclei involved in the regulation of the gonadotropic axis. These data were 314
confirmed by unchanged hormonal levels and weights of androgeno-dependent tissues.
315
316
4. Discussion 317
The present study shows that co-exposure of adult male mice to a DEHP/NP mixture induced 318
behavioral alterations, mainly at the lowest dose tested of 0.5 µg/kg/day. This dose did not trigger any 319
changes when each compound was tested separately. Moreover, the behavioral modifications induced 320
by each compound alone, seen in previous studies at the dose of 5 µg/kg/day, were no longer 321
observable with the mixture of the two products.
322 323
Male mice exposed to the DEHP/NP mixture at a dose of 0.5 µg/kg/day did not exhibit a 324
preference for receptive females. Such alterations can probably explain the lower number of male 325
mice in the treatment group emitting ultrasonic vocalizations, since this behavior is induced by 326
olfactory stimulation following female anogenital investigation. Despite an altered olfactory 327
preference, male mice exposed to the DEHP/NP mixture at 0.5 µg/kg/day initiated mating. The 328
latencies to initiate the first mount, and intromission, and to reach ejaculation, were not statistically 329
significant despite an increased tendency of these events to be increased in this exposed group. In 330
contrast, the frequency of behavior was significantly affected, as evidenced by a lower total number of 331
mounts and thrusts. These behavioral alterations could not be explained by changes in locomotor 332
activity or anxiety-state levels. Indeed, male mice exposed to a DEHP/NP mixture at 0.5 µg/kg/day 333
showed an increased activity and normal anxiety-state level. Altogether, our data point out a sexual 334
alteration induced by chronic exposure of adult male mice to a DEHP/NP mixture at 0.5 µg/kg/day.
335
Behavioral alterations induced by the DEHP/NP mixture may be caused by an endocrine disrupting 336
mode of action involving i) changes in circulating levels of gonadal testosterone, ii) interference with 337
hormone-receptor binding, or iii) modifications in the expression levels of sex steroid receptors in 338
neural areas that underlie male behavior. No changes were observed in testosterone levels, as was 339
confirmed by the unaltered weight of androgeno-dependent tissues. In agreement with this, analysis of 340
the number of kisspeptin-immunoreactive cells in the two hypothalamic areas involved in the 341
regulation of the gonadotropic axis revealed no differences between the three treatment groups. In 342
addition, the number of AR- and ERα-immunoreactive cells in the neural circuitry that underlies
343
sexual behavior was comparable between the three treatment groups. Therefore, one possibility could 344
be that chronic exposure to the DEHP/NP mixture at 0.5 µg/kg/day interfered with hormone-receptor 345
binding, although this hypothesis remains difficult to demonstrate clearly in vivo. Alternatively, the 346
behavioral changes could also be triggered by mechanisms that are not part of an endocrine disrupting 347
mode of action.
348 349
A common feature of exposure to one (DEHP or NP) or two (DEHP and NP) molecules is the lack 350
of effects on the gonadotropic axis. Indeed, no changes were seen in circulating levels of testosterone 351
or kisspeptin in the preoptic area or arcuate nucleus, whatever the dose of pollutants used (Capela et 352
al., 2018; Dombret et al., 2017; present study). We have previously discussed the fact that the 353
hypothalamus-pituitary-gonad axis seems less vulnerable in adults to exposure to low doses of DEHP 354
alone (Dombret et al., 2017) or NP alone (Capela et al., 2018). The present data show that exposure to 355
low doses of the DEHP/NP mixture does not affect this axis.
356
At the behavioral level, the majority of changes induced by exposure to the DEHP/NP mixture were 357
observed at a dose of 0.5 µg/kg/day. When used separately, neither DEHP nor NP could affect male 358
behavior at this dose. It is possible that a synergistic effect occurred in the neural circuitry that 359
underlies sexual behavior when the two substances were combined. Another interesting finding is that 360
exposure to the DEHP/NP mixture at 5 µg/kg/day did not induce any sexual alteration. DEHP and NP 361
have been respectively described for their anti-androgenic and estrogenic activities. When male mice 362
were exposed to each compound alone, the observed effects were not comparable. Firstly, exposure to 363
DEHP lengthened the motivational phase by affecting the emission of ultrasonic vocalizations and 364
male attractiveness (Dombret et al., 2017), while NP increased the emission of ultrasonic vocalizations 365
and the time from the first mount to ejaculation. There was a significant increase in the number of 366
mounts, intromissions and thrusts (Capela et al., 2018). Secondly, DEHP-induced effects could mainly 367
be attributed to an anti-androgenic activity through down-regulation of the neural AR without any 368
effect on ERα expression (Dombret et al., 2017), whereas NP exposure brought to mind the effects 369
reported for estradiol administration to adult rodents (Retana-Márquez et al., 2016). It was associated
370
with changes in the expression levels of both receptors in the neural circuitry underlying sexual 371
behavior (Capela et al., 2018). Whether anti-androgenic or estrogenic, each compound alone was able 372
to induce sexual alterations, suggesting that a physiological balance between both signaling pathways 373
is necessary to elicit normal behavior. Indeed, the AR and ERα signaling pathways play a 374
complementary role in the activation of male sexual behavior. When they are combined, it is possible 375
that these anti-androgenic and estrogenic substances exert antagonistic effects, which may explain the 376
absence of effects on male behavior. A comparable observation was reported in pre-pubertal male rats, 377
where exposure to a mixture of DBP and NP exerted antagonistic effects on LH and FSH levels in 378
comparison to each compound alone (Hu et al., 2014b).
379 380
5. Conclusion 381
The present work shows that exposure of adult male mice to a mixture of DEHP and NP affects 382
their olfactory preference, emission of ultrasonic vocalizations, and frequency of behavioral events 383
during mating. The induced changes in sexual behavior, together with increased locomotor activity, 384
were observed at the lowest dose tested (0.5 µg/kg/day). This did not trigger changes when male mice 385
were exposed to each compound separately, suggesting a synergistic effect of co-exposure to the two 386
compounds. The data obtained could be extremely relevant for several vertebrate species that use 387
olfactory cues and ultrasonic vocalization to detect and attract their female partner. A study in humans 388
reported an association between the levels of urine DEHP and NP metabolites and hypospadias in 389
boys (Choi et al., 2012). This indicates that combined exposure to these molecules can trigger adverse 390
effects. Whether such exposure also induces sexual or erectile deficiencies during adulthood remains 391
to be investigated. The results also show that behavioral effects, which were reported for each 392
molecule when used alone at the dose of 5 µg/kg/day (Capela et al., 2018; Dombret et al., 2017), were 393
no longer observable upon co-exposure, suggesting antagonistic effects of the two molecules. Overall, 394
despite the use of only two molecules, the present data highlight and confirm the need to assess 395
environmental endocrine disrupters in the context of combined exposure.
396
397
Acknowledgements 398
This work was supported by the “Agence Nationale de Sécurité Sanitaire de l'Alimentation, de 399
l'Environnement et du Travail” (Anses; Project n° 2012-2-077). We thank the INRA hormonal assay 400
platform for testosterone assays and the UPMC and INRA (UEPAO) platforms for taking care of the 401
animals.
402
403
References 404
Barakat R., Lin PP., Rattan S., Brehm E., Canisso IF., Abosalum ME., Flaws JA., Hess R., Ko C., 405
2017. Prenatal exposure to DEHP induces premature reproductive senescence in male mice.
406
Toxicol. Sci. 156, 96-108.
407
Bean NJ., 1982. Olfactory and vomeronasal mediation of ultrasonic vocalizations in male mice.
408
Physiol. Behav. 28, 31-37.
409
Buñay J., Larriba E., Moreno RD., Del Mazo J., 2017. Chronic low-dose exposure to a mixture of 410
environmental endocrine disruptors induces microRNAs/isomiRs deregulation in mouse 411
concomitant with intratesticular estradiol reduction. Sci. Rep. 7, 3373.
412
Capela D., Dombret C., Poissenot K., Poignant M., Malbert-Colas A., Franceschini I., Keller M., 413
Mhaouty-Kodja S., 2018. Adult male mice exposure to nonylphenol alters courtship vocalizations 414
and mating. Sci. Rep. 8, 2988.
415
Choi H., Kim J., Im Y., Lee S., Kim Y., 2012. The association between some endocrine disruptors and 416
hypospadias in biological samples. J. Environ. Sci. Health A. Tox. Hazard Subst. Environ. Eng. 47, 417
2173-2179.
418
Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 amending 419
Directives 2000/60/EC and 2008/105/EC as regards priority substances in the field of water policy.
420
Available at: https://www.ecolex.org/details/legislation/directive-201339eu-of-the-european- 421
parliament-and-of-the-council-amending-directives-200060ec-and-2008105ec-as-regards-priority- 422
substances-in-the-field-of-water-policy-lex-faoc127344/.
423
Dizinno G., Whitney G., 1977. Androgen influence on male mouse ultrasounds during courtship.
424
Horm. Behav. 8, 188-192.
425
Dombret C., Capela D., Poissenot K., Parmentier C., Bergsten E., Pionneau C., Chardonnet S., 426
Grange-Messent V., Keller K., Franceschini I., Mhaouty-Kodja S., 2017. Neural mechanisms 427
underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in 428
mice. Environ. Health Perspect. 125, 097001.
429
Hu Y., Li D-‐M., Han X-‐D., 2012. Analysis of combined effects of nonylphenol and Monobutyl 430
phthalate on rat Sertoli cells applying two mathematical models. Food Chem. Toxicol. 50, 457-463.
431
Hu Y., Wang R., Xiang Z., Qian W., Han X., Li D., 2014a. Mixture effects of nonylphenol and di-n- 432
butyl phthalate (monobutyl phthalate) on the tight junctions between Sertoli cells in male rats in 433
vitro and in vivo. Exp. Toxicol. Pathol. 66, 445-454.
434
Hu Y., Wang R., Xiang Z., Qian W., Han X., Li D., 2014b. Antagonistic effects of a mixture of low- 435
dose nonylphenol and di-n-butyl phthalate (monobutyl phthalate) on the Sertoli cells and serum 436
reproductive hormones in prepubertal male rats in vitro and in vivo. PLoS One. 9, e93425.
437
Kauffman AS., Gottsch ML., Roa J., Byquist AC., Crown A., Clifton DK., Hoffman GE., Steiner RA., 438
Tena-Sempere M., 2007. Sexual differentiation of Kiss1 gene expression in the brain of the rat.
439
Endocrinology 148, 1774-1783.
440
Laws S.C., Carey S.A., Ferrell J.M., Bodman G.J., Cooper R.L., 2000. Estrogenic activity of 441
octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicol. Sci. 54, 154-167.
442
Li D., Hu Y., Shen X., Dai X., Han X., 2010. Combined effects of two environmental endocrine 443
disruptors nonyl phenol and di-n-butyl phthalate on rat Sertoli cells in vitro. Reprod. Toxicol. 30, 444
438-445.
445
Naulé L., Marie-Luce C., Parmentier C., Martini M., Albac C, Trouillet A-C., Keller M., Hardin- 446
Pouzet H., Mhaouty-Kodja S., 2016. Revisiting the neural role of estrogen receptor beta in male 447
sexual behavior by conditional mutagenesis. Horm. Behav. 80, 1-9.
448
Navarro VM., Gottsch ML., Wu M., García-Galiano D., Hobbs SJ., Bosch MA., Pinilla L, Clifton 449
DK., Dearth A., Ronnekleiv OK., Braun RE., Palmiter RD., Tena-Sempere M., Alreja M., Steiner 450
RA., 2011. Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the 451
arcuate nucleus of the male mouse. Endocrinology 152, 4265-4275.
452
Nyby J., Wysocki CJ., Whitney G., Dizinno G., 1977. Pheromonal regulation of male mouse 453
ultrasonic courtship (Mus musculus). Anim. Behav. 25, 333-341.
454
Ogawa S., Lubahn DB., Korach KS., Pfaff DW., 1997. Behavioral effects of estrogen receptor gene 455
disruption in male mice. Proc. Natl. Acad. Sci. U.S.A. 94, 1476-1481.
456
Ogawa S., Washburn TF., Taylor J., Lubahn DB., Korach KS., Pfaff DW., 1998. Modifications of 457
testosterone-dependent behaviors by estrogen receptor-alpha gene disruption in male mice.
458
Endocrinology 139, 5058-5069.
459
Pocar P., Fiandanese N., Secchi C., Berrini A., Fischer B., Schmidt JS., Schaedlich K., Borromeo V., 460
2012. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long- 461
term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology 153, 462
937-948.
463
Pomerantz SM., Nunez AA., Bean NJ. 1983. Female behavior is affected by male ultrasonic 464
vocalizations in house mice. Physiol. Behav. 31, 91-96.
465
Raskin K., de Gendt K., Duittoz A., Liere P., Verhoeven G., Tronche F., Mhaouty-Kodja S., 2009.
466
Conditional inactivation of androgen receptor gene in the nervous system: effects on male 467
behavioral and neuroendocrine responses. J. Neurosci. 29, 4461-4470.
468
Retana-Márquez S., Juárez-Rojas L., Hernández A., Romero C., López G., Miranda L., Guerrero- 469
Aguilera A., Solano F., Hernández E., Chemineau P., Keller M., Delgadillo JA., 2016. Comparison 470
of the effects of mesquite pod and Leucaena extracts with phytoestrogens on the reproductive 471
physiology and sexual behavior in the male rat. Physiol. Behav. 164, 1-10.
472
Ruka KA., Burger LL., Moenter SM., 2016. Both estrogen and androgen modify the response to 473
activation of neurokinin-3 and κ-opioid receptors in arcuate kisspeptin neurons from male mice.
474
Endocrinology 157, 752–763.
475
Smith JT., Dungan HM., Stoll EA., Gottsch ML., Braun RE., Eacker SM., Clifton DK., Steiner RA., 476
2005. Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male 477
mouse. Endocrinology 146, 2976-2984.
478
Wersinger SR., Sannen K., Villalba C., Lubahn DB., Rissman EF., De Vries GJ., 1997. Masculine 479
sexual behavior is disrupted in male and female mice lacking a functional estrogen receptor α gene.
480
Horm. Behav. 32, 176-183.
481
482
Figure legends 483
Figure 1. Effects of adult exposure to a DEHP/NP mixture on male sexual behavior. A. Total time 484
spent in the chemo-investigation of male and sexually receptive female stimuli by males exposed to 485
the vehicle (Veh) or DEHP/NP mixture (0.5 or 5 μg/kg/day). B. Number of entries into the male or 486
female arm of the Y maze. C. Discrimination index expressed as time spent investigating the female 487
minus time spent investigating the male divided by the total time of chemo-investigation. Data are 488
expressed as the means ± S.E.M. of 9-10 males per treatment group. One-way ANOVA (
ap < 0.001) 489
demonstrated an effect of exposure; post hoc analyses showed differences (p < 0.001) between males 490
exposed to the mixture at 0.5 µg/kg/day and the other two groups. D. Percentage of males emitting 491
ultrasonic vocalizations in the presence of a sexually receptive female. Chi square test analysis showed 492
a statistically significant difference (*p < 0.05). E-F. Latency to the first mount, intromission, and to 493
ejaculation (E) and duration of mating expressed as the time between the first mount and ejaculation 494
(F) for males exposed to the vehicle (Veh) or a DEHP/NP mixture at 0.5 or 5 μg/kg/day. G. Number 495
of mounts (left), intromissions (middle) and thrusts (right) exhibited by males. One-way ANOVA (a) 496
showed an effect of exposure; *p < 0.05 versus the vehicle group.
497 498
Figure 2. Anxiety and locomotor activity were affected by exposure to a DEHP/NP mixture. A.
499
Left. Spontaneous activity by males exposed to the vehicle (Veh) or to DEHP/NP at 0.5 or 5 500
μg/kg/day measured for 140 min. *p < 0.05 or **p < 0.01, post hoc analyses showing a higher activity 501
for the DEHP/NP-0.5 group by comparison to males exposed to DEHP/NP-5 at 80 and 100 min of the 502
test. Right. Cumulative activity recorded for the three treatment groups during the 140 min of the test.
503
Data are expressed as the means ± S.E.M. of 9-10 males per treatment group;
ap < 0.05 general effect 504
of exposure. B. Anxiety-state level measured in the zero maze paradigm. Latency to the first entry into 505
the open arm (Left), the number of entries into the open arm (middle) and time spent in the open arms 506
(right) were analyzed. Data are expressed as the means ± S.E.M. of 9-10 males per treatment group.
507
One-way ANOVA (a) showed an effect of exposure to DEHP/NP. *Post hoc analyses showed 508
significant differences between DEHP/NP-5 and vehicle groups.
509
510
Figure 3. Kisspeptin immunoreactivity and hormonal levels are unchanged following chronic 511
exposure to a DEHP/NP mixture. Mice were exposed to the vehicle or DEHP/NP mixture at 0.5 or 5 512
µg/kg/day. A. Upper panel: representative immunolabeling in the periventricular nuclei is shown 513
(left), with increased magnification of a kisspeptin-immunoreactive cell body (right). Lower panel:
514
quantification of the number of kisspeptin-immunoreactive neurons and fiber density in the 515
anteroventral periventricular (AVPV) and periventricular (PeN) nuclei. B. Representative 516
immunolabeling in the arcuate nucleus (upper panel) and quantification of fiber density (lower panel).
517
Data are expressed as the means ± S.E.M. of 6 males per treatment group. Scale bar = 20 µm (A-right 518
panel) and 50 µm (A-left panel and B). C-E. Circulating levels of testosterone (C) and weight of 519
seminal vesicles (SV; D) and testes (E) are expressed as a percentage of body weight (bw). Data are 520
expressed as the means ± S.E.M. of 9-10 males per treatment group.
521
522
Figure 4. The number of androgen receptor (AR)-immunoreactive (ir) cells was unchanged upon 523
exposure to a DEHP/NP mixture. A. Representative AR-ir cells in the posterodorsal medial 524
amygdala (MeA), bed nucleus of stria terminalis (BNST) and medial preoptic nucleus (MPN) of males 525
exposed to the vehicle (Veh) or a DEHP/NP mixture at 0.5 or 5 µg/kg/day. B. Quantitative analyses of 526
the number of AR-ir neurons in chemosensory areas. Data are expressed as the means ± S.E.M. of 6 527
males per treatment group. Scale bar, 100 µm.
528 529
Figure 5. The number of α estrogen receptor (ERα )-immunoreactive (ir) cells was unchanged 530
upon exposure to the DEHP/NP mixture. A. Representative ERα-ir cells in the posterodorsal medial 531
amygdala (MeA), bed nucleus of stria terminalis (BNST) and medial preoptic nucleus (MPN) of males 532
exposed to the vehicle (Veh) or a DEHP/NP mixture at 0.5 or 5 µg/kg/day. B. Quantitative analyses of 533
the number of ERα-ir neurons in chemosensory areas. Data are expressed as the means ± S.E.M. of 6 534
males per treatment group. Scale bar, 100 µm.
535
536
537 538
Figure 1
0 0,1 0,2 0,3 0,4 0,5
1"
Veh 0.5 5 DEHP/NP (µg/kg/d)
A
Discrimination index
***
B
0"
10"
20"
30"
40"
50"
60"
70"
1"
% vocalizing males
a
***
0"
10"
20"
30"
40"
50"
60"
1" 2" 3"
Latency to behavior (min)
Mount Intromission Ejaculation
D
Mating duration (min)
E
0 5 10 15 20
1" 2" 3"
0 20 40 60 80
1"
Total chemoinvestigation time (s) Number of entries
F C
0 2 4 6 8 10
1"
0 5 10 15 20 25
1"
Number of mounts Number of MI Number of thrusts
G
Male Female
Veh DEHP/
NP-0.5 DEHP/
NP-5
a
*
0 100 200 300 400 500 600
1"
a
* 0 5 10 15 20 25
1"
*
539 540
Figure 2
Total locomotor activity0 500 1000 1500 2000
1"
a
Number of entries Time spent in open arms (s)
B
0 2 4 6 8 10
1"
0 10 20 30 40 50
1"
* *
0 100 200 300 400
1" 2" 3" 4" 5" 6" 7"
Locomotor activity per 20 min
A
Veh 0.5 5 DEHP/NP (µg/kg/d)
Latency (s)
0"
100"
200"
300"
400"
1"
20 40 60 80 100 120 140
Time (min)
a a
*
**
541 542
Figure 3
0 2 4 6 8
AVPV PeN
0 1 2 3 4 5
AVPV PeN
0 5 10 15 20
ARC
Fiber density (AU)
Fiber density (AU)
Cell number
B A
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
1"
0,0 0,5 1,0 1,5 2,0 2,5
1"
Testosterone (ng/ml) SV weight (% bw) C
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7
1"
Testis weight (% bw)
D E
Veh 0.5 5 DEHP/NP (µg/kg/d)
543 544
Figure 4
Veh DEHP/NP-0.5 DEHP/NP-5
AR-ir cell number MeABNSTMPN A
B
0 100 200 300 400
1"
0 500 1000 1500 2000
1"
0 200 400 600 800 1000 1200
1"
MeA BNST MPN
Veh 0.5 5 DEHP/NP (µg/kg/d)
545 546
Figure 5
ERα-ir cell number
B
Veh DEHP/NP-0.5 DEHP/NP-5
A
MeABNSTMPN
0 200 400 600 800 1000 1200 1400
1"
0 500 1000 1500 2000 2500 3000
1"
0 100 200 300 400 500
1"
MeA BNST MPN
Veh 0.5 5 DEHP/NP (µg/kg/d)
547 548
Table 1. Summary of neuroendocrine and behavioral effects induced by adult exposure to DEHP 549
alone, NP alone or combined exposure to both molecules in male mice.
550
Molecules DEHP
(Dombret et al., 2017)
NP (Capela et al., 2018)
DEHP/NP mixture (present study)
Doses 0.5 µg/kg/day 5 µg/kg/day 0.5 µg/kg/day 5 µg/kg/day 0.5 µg/kg/day 5 µg/kg/day
Behavioral effects Sexual behavior
Olfactory preference Unaffected Unaffected Unaffected Unaffected No preference Unaffected
Ultrasonic vocalizations Unaffected
Unaffected number and duration, altered ratio
of USVs syllables
Unaffected Increased number
and duration Few males vocalized Unaffected
Latency to intromission, and
ejaculation Unaffected
Increased latency to intromission
and ejaculation Unaffected Increased latency
to ejaculation No significant effect Unaffected
Number of mounts, intromissions
and thrusts Unaffected Unaffected Unaffected Increased number of
mounts, intromissions and thrusts
Reduced number of mounts and thrusts
Unaffected
General behaviors
Locomotor activity Unaffected Unaffected Unaffected Unaffected Increased Unaffected
Anxiety-state level Unaffected Unaffected Unaffected Increased Unaffected Reduced
Chemosensory areas Number of androgen receptor (AR)- and estrogen receptor (ER)α−immunoreactive cells
Medial amygdala Unaffected Reduced for AR Unaffected Reduced for AR
Increased for ERα Unaffected Unaffected Bed nucleus of stria terminalis Unaffected Reduced for AR Unaffected Increased for AR Unaffected Unaffected
Medial preoptic nucleus Unaffected Reduced for AR Unaffected Reduced for AR Unaffected Unaffected
Number of kisspeptin-immunoreactive cells
Medial preoptic nucleus Unaffected Unaffected Unaffected Unaffected Unaffected Unaffected
Arcuate nucleus Unaffected Unaffected Unaffected Unaffected Unaffected Unaffected
